Contiguous capillary electrospray sources and analytical devices

ABSTRACT

Contiguous capillaries useful for separating and electrospraying a fluid comprising analyte and electrolyte are provided. The contiguous capillaries have spray tips at one end of the capillaries and electrically conductive portions in proximity to the spray tips. Methods for making the contiguous capillaries and their use as electrospray sources are also disclosed. Apparatus and methods for conveying analyte ions from the capillaries into analytical instruments, such as a mass spectrometer, are also disclosed. The disclosed contiguous capillaries may be used to carryout electrophoresis separation and electrospray ionization of analytes. Methods for obtaining the mass spectra of macromolecular analytes at concentrations lower than previously possibly are provided using the apparatus and procedures described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2003/033200, filed Oct. 20, 2003, which claims the benefit of U.S.Provisional Application No. 60/420,003, filed Oct. 21, 2002, thedisclosure of which is incorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

The work leading to the disclosed inventions was finded in whole or inpart with Federal funds from the National Cancer Institute, NationalInstitutes of Health, under Contract No. NO1-CO-12400. Accordingly, theU.S. Government has rights in these inventions.

FIELD OF THE INVENTION

The present inventions are related to the field of molecular analysis offluids comprising analyte and electrolyte using capillaries. Related areinventions for devices and methods for electrospraying analyte ions fromcapillaries into analytical instruments, such as a mass spectrometer.The capillaries may be used in the electrophoresis separation of, and inthe electrospraying of analytes.

BACKGROUND OF THE INVENTION

Mass spectrometry (MS) is arguably among the most useful detectionschemes for capillary electrophoresis (CE) and high performance liquidchromatography (HPLC) largely due to the limited information obtainedwith other common detection techniques, such as UV, visible, andfluorescence spectrometry. The limits of these common detectiontechniques are particularly evident in the identification and analysisof macromolecules, such as peptides and proteins. While CE is itselfused as an analytical method, CE has also been utilized to separateanalytes prior to analysis by both fast atom bombardment (Moseley, M.A., Deterding, L. J., Tomer, K. B., Jorgenson, J. W., J. Chromatog.,1989, 480, 197) and matrix assisted laser desorption ionization MSinstrumentation (Preisler, J., Foret, F., Karger, B. L., Anal. Chem.,1988, 70, 5278). In addition, a very useful technique is obtained byinterfacing CE directly online with electrospray ionization (ESI) massspectrometry, referred to as “CE-ESI-MS”.

The successful operation of a CE-ESI-MS system typically requires aclosed circuit for both the CE separation and the electrosprayionization processes. Three major designs have been advanced, namely,coaxial sheath flow (Smith, R. D., Barinaga, C. J., Udseth, H. R., Anal.Chem., 1988, 60, 1948), liquid junction (Lee, E. D., Muck, W., Henion,J. D., Covey, T. R., Biomed. Environ. Mass. Spectrom., 1989, 18, 844),and sheathless flow (Olivares, J. A., Nguyen, N. T., Yonker, C. R.,Smith, R. D., Anal. Chem., 1987, 59, 1230). Coaxial sheath flow is thebasis of most commercial instruments, though it suffers in sensitivity.The low sensitivity of a coaxial sheath flow CE design arises largelyfrom the relatively high sheath flow compared to the flow from the CEcapillary, resulting in not only a large dilution of the elutinganalytes, but also in hindered desorption of ions due to the non-optimalelectrospray that results at such high flow rates. Coupling CE onlinewith MS through a liquid junction arrangement requires tedious capillaryalignment and end-to-end butting of the separation capillary and thespray tip. Unfortunately, even under the best conditions, sensitivity iscompromised by loss and spreading of sample analytes in the relativelylarge dead volume of the liquid junction.

Electrolytic interfaces for coupling electrophoresis capillaries toelectrospray tips (spray tips) have been designed for conveying analyteions to mass spectrometers. Such interfaces have been designed to effectcompletion of an electrolytically conductive fluid circuit in thecapillary tube, which include openings near the spray tip, and physicalbreaks connected by permeable sheaths near the spray tip. Unfortunately,these breaks and openings result in analyte loss, disruption of thefluid flow path, disruption of the electric field, or a combination ofthese effects near the spray tip, which ultimately degrades massdetection sensitivity.

Sheathless flow is, in principle, a desirable design for coupling CEonline with MS, one reason being that analyte dilution is minimizedcompared to capillaries incorporating a sheathed design. A variety ofsheathless designs have been described that satisfy the requirement ofclosing the CE separation capillary circuit while simultaneouslyproviding an electrical potential to the spray tip. These, for example,include the use of a single capillary whereby electrical contact isestablished through: (a) coating the capillary outlet with a conductivemetal (Olivares, J. A., Nguyen, N. T., Yonker, C. R., Smith, R. D.,Anal. Chem., 1987, 59, 1230; Kelly, J. F, Ramaley, L, Thibault, P.,Anal. Chem., 1997, 69, 51; Barraso, M. B., deJong, A. P., J. Am. Soc.Mass Spectrom., 1999, 10, 1271; Chang, Y. Z., Her, G. R., Anal. Chem.,2000, 72, 626; Figeys, D., Oostveen, I., Ducert, A., Aebersold, R.,Anal. Chem., 1996, 68, 1822; Kriger, M. S., Cook, K. D., Ramsey, R. S.,Anal. Chem., 1995, 67, 385; Wilm, M., Mann, M., Anal. Chem., 1996,68, 1) or polymer (Maziarz, E. P., Lorentz, S. A., White, T. P., Wood,T. D., J. Am. Soc. Mass Spectrom., 2000, 11, 659); (b) insertion of aconductive wire into the outlet of the capillary (Fang, L., Zhang, R.,Williams, E. R., Zare, R. N., Anal. Chem., 1994, 66, 3696) or through asmall pinhole in the wall of the capillary (Cao, P., Moini, M., J. Am.Soc. Mass Spectron., 1998, 9, 1081; Smith, A. D., Moini, M., Anal.Chem., 2001, 73, 240); (c) splitting the capillary effluent at or nearthe capillary outlet to fill the gap between the capillary and an outercoaxial metallic sleeve (Moini, M. Anal. Chem. 2001, 73, 3497,Petersson, M. A., Hulthe, G., Fogelqvist, E., J. Chromatogr. A, 1999,854, 141), or (d) adjusting the position of the outlet of the capillarysuch that electrical contact is established through the air to thegrounded inlet capillary of the MS (Mazereeuw, M., Hofte, A. J. P.,Tjaden, U. R., van der Greef, J., Rapid Commun. Mass Spectrum., 1997,11, 981). Another strategy for the fabrication of a sheathless interfaceis to use two pieces of capillary whereby the CE capillary is connectedto a short spray tip via a sleeve. The sleeve could be a piece ofmicrodialysis tubing (Severs, J. C., Smith, R. D., Anal Chem., 1997, 69,2154), stainless steel tubing (Figeys, D., Ducret, A., Yates, J. R.,Aebersold, R., Nature Biotechnol., 1996, 14, 1579), or a micro-tee(Tong, W., Link, A., Eng, J. K., Yates, J. R., Anal. Chem., 1999, 71,2270). Although these approaches do produce operational interfaces,their fabrication requires delicate manipulation of miniaturizedcomponents and they suffer in their robustness. In the two pieceapproach, the CE separated zones are invariably broadened at thejunction between the separation column and the tip, since the insidediameter of the sleeve has to be larger than the outside diameter of theseparation capillary. Furthermore, these junctions often suffer frommisalignment and imperfect butting of the two pieces of capillary.Single capillary methods appear to disrupt the CE separation the least,however, metal coatings on fused silica capillaries are not durable anddrilling pin-holes through capillary walls is a delicate andirreproducible procedure. Once operational, a split-flow interface ishighly sensitive, as demonstrated by Moini et al. who reported theseparation and detection of proteins from human red blood cells atattomole levels (Moini, M., Demars, S. M., Huang, H., Anal. Chem., 2002,74, 3772). Sheathless interfacing has been the subject of several recentreviews, where the advantages and limitations of this design have beenenumerated (Tong, W., Yates, J. R., Chromatographia Supplement, 2001,53, S90; Ding, J., Vouros, P., Anal. Chem., 1999, 71, 378A; Gelpi, E.,J. Mass Spectrum., 2002, 37, 241; Moini, M., Anal. and Bioanal. Chem.,2002, 373, 466).

Thus, there is a need to provide improved capillary designs for couplingCE with MS that overcome these problems.

SUMMARY OF THE INVENTION

The inventions described herein enable one to obtain the mass spectra ofmacromolecular analytes, such as peptides, proteins, RNA, DNA,oligonucleotides, and polymers at concentrations lower than previouslypossibly. As provided herein, the present invention achieves one goal ofproviding new capillary designs that directly couple CE online with MS.New contiguous capillaries are provided that do not require a sheathedopening or break in the capillary near the spray tip. The new contiguouscapillaries are not only rugged and simple in design, but they alsoeffect an increase in macromolecular analyte detection sensitivity of upto about 100-fold in CE-ESI-MS devices—a veritable quantum leap inmolecular detection technology.

Thus, in a first aspect of the present invention, there are providedcontiguous capillaries for electrospraying a fluid comprising analyteand electrolyte. In this aspect, each of the capillaries includes aninlet end to supply the fluid into the capillary, a spray tip forspraying fluid out of the capillary, and an electrically conductiveportion of the capillary in proximity to the spray tip. In this aspectof the invention, the fluid containing the analyte enters the inlet endand exits the spray tip. Also, the electrically conductive portion isdesigned to minimize analyte loss while maintaining electricalconductivity.

In another aspect of the present invention, there are providedcontiguous capillaries that are suitable for conveying fluid samplescontaining analytes into an analytical instrument. In this aspect of theinvention, the contiguous capillaries include an inlet end to supply thefluid into the capillary, a spray tip for spraying fluid out of thecapillary, and an electrically conductive portion of the capillary inproximity to the spray tip. In this aspect of the invention, the wall ofthe electrically conductive portion of the capillary is capable ofblocking passage of analyte molecules therethrough.

In another aspect of the present invention there are providedelectrospray sources, each including a contiguous capillary forseparating and electrospraying a fluid comprising analyte andelectrolyte. Here, each of the capillaries includes an inlet end tosupply the fluid into the capillary, a spray tip for spraying fluid outof the capillary, and an electrically conductive portion of thecapillary in proximity to the spray tip. In this aspect of theinvention, the fluid containing the analyte enters the inlet end andexits the spray tip. The electrically conductive portion may provide avoltage along the capillary interior, at the spray tip, or both. Theelectrically conductive portion is designed to minimize analyte losswhile maintaining electrical conductivity.

In another aspect of the present invention there are providedapparatuses for conveying analyte ions into an analytical instrument. Inthis aspect, each apparatus includes a contiguous capillary having aelectrically conductive portion near its spray tip, an electrodeexterior to the electrically conductive portion, a spraycounter-electrode in proximity to the spray tip, and a power supplyconnected to the electrode and the spray counter-electrode to provide aspray voltage.

In various aspects of the invention, each capillary includes: an inletend to supply a fluid into the capillary, the fluid comprising analyteand electrolyte; a spray tip to spray fluid out of the end of thecapillary that is opposite to the inlet end; and an electricallyconductive portion of the capillary in proximity to said spray tip. Theelectrically conductive portion may provide a voltage along thecapillary interior, at the spray tip, or both. Also, the electricallyconductive portion is designed to minimize analyte loss whilemaintaining electrical conductivity.

Also in various aspects of the invention, the electrode exterior to theelectrically conductive portion is in electrically conductive contactwith the fluid interior to the electrically conductive portion of thecapillary. The spray counter-electrode is provided in proximity to thespray tip, and includes an opening in fluid communication with theanalytical instrument. The power supply, which is connected to theelectrode and the spray counter-electrode provides a spray voltage forgenerating an electrospray comprising analyte ions. The spray voltageconveys at least a portion of the analyte ions through the opening andinto the analytical instrument.

In other aspects of the invention there are provided methods of maltinga contiguous capillary suitable for separating and electrospraying afluid comprising analyte and electrolyte. In these aspects, the methodsof making the capillaries include providing a capillary having an inletend and a spray tip, and etching a portion of the capillary wall inproximity to the spray tip to provide an electrically conductive portionof the capillary.

In other aspects of the present invention there are provided methods ofconveying a fluid comprising analyte and electrolyte into an analyticalinstrument. These methods typically include providing a contiguouscapillary, transporting the fluid through the contiguous capillary,providing an electrode exterior to the electrically conductive portion,providing a spray counter-electrode in proximity to the spray tip, andapplying a spray voltage between the electrode and the spraycounter-electrode. These aspects of the invention effect electrosprayionization of the analyte exiting the spray tip, so that at least aportion of the analyte enters the analytical instrument.

In these methods for conveying fluid into analytical instruments, eachcapillary includes: an inlet end to supply a fluid into the capillary,the fluid comprising analyte and electrolyte; a spray tip to spray fluidout of the end of the capillary that is opposite the inlet end; and anelectrically conductive portion of the capillary in proximity to thespray tip. The electrically conductive portion provides a voltage alongthe capillary interior, at the spray tip, or both. Also, theelectrically conductive portion is designed to minimize analyte losswhile maintaining electrical conductivity.

Also in these methods, the electrode exterior to the electricallyconductive portion is in electrically conductive contact with the fluidinterior to the capillary. The spray counter-electrode being inproximity to the spray tip includes an opening in fluid communicationwith the analytical instrument. The power supply is connected to theelectrode and the spray counter-electrode to provide a spray voltage,which generates an electrospray comprising analyte ions. At least aportion of the analyte ions is typically conveyed through the opening inthe spray counter-electrode and into the analytical instrument.

In another aspect of the present invention, there are provided methodsof obtaining the mass spectra of analyte molecules. In these methods,the mass spectra are obtained by providing a fluid comprising analyteand electrolyte, providing one of the contiguous capillaries of anaspect of the present invention described herein, transporting the fluidthrough the contiguous capillary, providing an electrode exterior to theelectrically conductive portion, providing a spray counter-electrode inproximity to the spray tip for producing an electrospray comprisinganalyte ions. A spray voltage is applied between the electrode and thespray counter-electrode to effect electrospray ionization of the analyteexiting the spray tip, so that at least a portion of the analyte ionsenters a mass spectrometer through the opening, in which the m/z valuesof the analyte ions are measured to provide the mass spectra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is cross-sectional view of one embodiment of a contiguouscapillary of the present invention. Dimensions are not drawn to scale inthis or other schematic drawings.

FIG. 2A is a perspective view of one embodiment of a electrospray sourceof the present invention.

FIG. 2B is a top view of the embodiment of a electrospray source in FIG.2A looking along line I-I.

FIG. 3A is a perspective view of one embodiment of a electrospray sourceof the present invention.

FIG. 3B is a top view of the embodiment of a electrospray source in FIG.3A looking along line I-I.

FIG. 4 is a photograph of a contiguous capillary electrospray sourcemounted on a LCQ-DECA MS nanoelectrospray assembly, as described in theexamples.

FIG. 5 shows the CE-ESI-tandem MS results of 25 femtomole of[Glu1]-Fibrinopeptide B according to the examples:

-   -   A. base-peak capillary electrophoresis electropherogram;    -   B. full scan mass spectrum of the [M+2H]2+[Glu1]-fibrinopeptide        B molecular ion; and    -   C. tandem MS fragment spectrum of the m/z 786.8 ion.

FIG. 6 is an electropherogram showing the separation of a mixture ofbioactive peptides according to the examples. The inset of FIG. 6 givesthe corresponding separation of the same peptide mixture at aconcentration of 25 mg/mL, using similar CE conditions.

FIG. 7 shows a schematic diagram of an electrospray source of thepresent invention (not drawn to scale).

FIG. 7 shows a schematic diagram of an electrospray source of thepresent invention.

FIG. 8 is a photograph of a contiguous capillary electrospray sourcemounted on a LCQ-DECA MS nanoelectrospray assembly, as described in theexamples.

FIG. 9 shows the CE-ESI-tandem MS of 25 femtomoles of[Glu¹]-Fibrinopeptide B;

-   -   A. base-peak electropherogram;    -   B. full scan mass spectrum of the [M+2H]²⁺[Glu¹]-fibrinopeptide        B molecular ion; and    -   C. tandem MS fragment spectrum of the m/z 786.8 ion. Column:        bare fused silica capillary, 60 cm×360 μm o.d.×75 μm i.d.×25 μm        tip i.d.; separation potential: 15 kV; observed CE current: 16        μA; buffer: 1 M acetic acid, pH=2.4; temperature: 22° C.; sample        concentration: 2 μM; injection time: 5 s at 0.5 psi (˜12.5 nL        total injection volume).

FIG. 10 shows the CE-ESI-tandem MS of a mixture of bioactive peptides.Experimental conditions: As in FIG. 7. sample concentration: 3 μg/mL;injection time: 10 s at 0.5 psi; solutes: 1=bradykinin; 2=substance P;3=bradykinin fragment 1-5; 4=[arg]-vasopressin; 5=luteinizing hormonereleasing hormone; 6=bombesin; 7=leucine enkephalin; 8=methionineenkephalin; 9=oxytocin. Inset: Corresponding CE separation of the samepeptide mixture at a concentration of 25 μg/mL.

FIG. 11 shows the CE-ESI-MS results of 100 fmoles of a tryptic digest ofhorse apomyoglobin. Experimental conditions: As in FIG. 7;

-   -   A. Base peak electropherogram;    -   B. molecular ion scan of the [M+2H]²⁺ ion at m/z=690.7; and    -   C. CID fragment ion spectrum of the [M+2H]²⁺ ion at m/z=690.7.        The CID spectrum was searched against the NCBI non-redundant        database using SEQUEST and identified as HGTVVLTALGGILK [SEQ ID        No: 3 ]with an Xcorr=4.95.

DETAILED DESCRIPTION

The coupling of capillary electrophoresis and mass spectrometry may becarried out using the contiguous capillaries of the present invention.Fluid samples containing analyte and electrolyte may be separated andelectrosprayed into an analytical device, such as a mass spectrometer,by using the contiguous capillaries of the present invention as providedherein. Almost any type of analyte molecule may be analyzed using thepresent inventions, including both small molecules and macromolecules.The analytes may range in molecular weight from about 20 g/mole up toabout 1,000,000 g/mol. Within this range, small molecules such asorganic synthetic chemicals, pharmaceuticals, and amino acids typicallyhave molecular weights within about the lower three orders of magnitudeof this range, whereas macromolecules such as proteins, nucleic acidsand polymers typically have molecular weights within about the upperthree orders of magnitude of this range. The analytes are typicallyprepared in solution with a suitable solvent and electrolyte. Suitablesolvents, which are typically capable of dissolving both electrolyte andanalyte, provide a fluid that is capable of being transported along theaxis within the interior of a capillary, e.g., water. Forces typicallyused to transport the fluid include hydrostatic forces, electrophoreticforces, electroosmosis forces, or a combination of two or more of theseforces.

Referring to FIG. 1, one embodiment of the present invention provides acontiguous capillary 10 for electrospraying a fluid comprising analyteand electrolyte (fluid not shown). The contiguous capillary 10 includesan inlet end 12 to supply the fluid into the capillary 14, a spray tip18 for spraying fluid out of the capillary at opening 20. Anelectrically conductive portion of the capillary 16 is positioned inproximity to the spray tip.

Capillaries used in the present invention may be made of almost anymaterial that can be formed into a thin tube. Typically the capillarymaterial is not electrically conductive, but a portion of the capillaryis made conductive by an etching process as provided below. Suitablecapillary materials include plastic and glass. Fused silica capillariesare typically used and are commercially available from PolymicroTechnologies, LLC. Fused silica capillaries having a protective coating,such as a polyimide coating, are desirable for controlling the locationof etching during preparation of the electrically conductive portion, asdiscussed below.

Suitable capillaries used in the present invention are available in avariety of dimensions. The length of the contiguous capillaries that maybe used in the present invention typically varies from about 5centimeters to about a meter, and is more typically in the range of fromabout 25 to about 75 centimeters. The inside diameter of the contiguouscapillaries is typically narrow to dissipate heat generated from ohmicheating caused by the application of a voltage on the fluid. The insidediameter is typically less than about 200 microns, which is suitable inuses when up to several kilovolts are applied along the length of thecapillary. The inside diameter of the contiguous capillaries typicallyranges from about 2 microns to about 100 microns, more typically fromabout 10 microns to about 75 microns, and even more typically from about20 microns to about 50 microns. Capillary wall thickness also varies,and is typically in the range of from 10 microns to 1 mm, more typicallyfrom 20 microns to 500 microns, and even more typically from 40 micronsto 250 microns.

The electrically conductive portion 16 is designed to minimize analyteloss through the wall while maintaining electrical conductivity. As usedherein, the term “electrically conductive” is intended to mean any modeby which electrons may be transported, including metallic conduction,electrolytic conduction, and inductive conduction. While not being boundto a particular theory of electrically conductivity, it is believed thatat least one or more of these modes causes electrical conductivity ofthe electrically conductive portion of the capillary.

In various embodiments of the present invention, the electricallyconductive portion includes regions in the capillary wall that aresufficiently thin to permit transport of electrons through the wall ofthe capillary. In these embodiments, the electrically conductive portionof the capillary typically has a wall thickness less than the wallthickness of the non-conductive capillary that is upstream from theelectrically conductive portion. In certain embodiments, theelectrically conductive portion of the capillary has a wall thicknessless than the wall thickness of the capillary adjacent to theelectrically conductive portion. Accordingly, the capillary wallthickness at the conductive portion is typically less than about 50microns, more typically less than about 40 microns, and even moretypically less than about 30 microns. The conductive portion typicallycomprises the capillary material. More typically, the conductive portionconsists essentially of the capillary material, such as fused silica orpolymer.

In various embodiments of the present invention, the electricallyconductive portion includes pores having a size to block passage ofanalyte while permitting passage of electrolyte therethrough. In theseembodiments one mode of electrical conductivity includes electrolyticconduction resulting from the passage of electrolyte through the pores.In certain embodiments, the pores permit passage of electrolyte ionshaving a molecular mass of less than about 300 g/mol.

In these embodiments, the degree by which the pores block passage of theanalytes depends on pore size and macromolecular size. Pores muchsmaller than the analyte size may completely block passage of theanalyte, while pores about the size of the analyte size may partiallyblock passage of the analytes. By “analyte size” as used herein refersto the hydrodynamic radius of a single analyte molecule or aggregate ofanalyte molecules in the fluid solution. In certain embodiments, thepores are able to partially block the passage of analytes having amolecular weight of greater than about 100 g/mol, typically greater thanabout 500 g/mol, and more typically greater than about 1,000 g/mol.

A suitable spray tip 18 used in the present inventions has an opening 20that is about the same diameter as, or smaller than the inside diameterof the capillary. A suitable spray tip opening 20 is typically smallerthan about 100 microns in diameter, more typically smaller than about 50microns in diameter, and even more typically smaller than about 30microns in diameter.

In the present invention, the spray tip 18 is proximate to theelectrically conductive section 16 of the capillary. In certainpreferred embodiments of the present invention, a suitable distancebetween the spray tip opening 20 and the electrically conductive section16 is typically greater than about 5 mm, more typically greater thanabout 10 mm, typically less than about 200 mm, more typically less thanabout 100 mm, and even more typically less than about 50 mm.

In certain embodiments of the present invention, the electricallyconductive portion of the capillary is fragile. In these embodiments, itis desirable to affix at least a portion of the capillary with theconductive portion within a support structure. In certain embodiments,the support structure is capable of holding a buffer solution. Incertain embodiments, the support structure may be used to contain anetching solution for preparing the electrically conductive portion asprovided herein.

The contiguous capillaries of the present invention are useful forcarrying out a wide variety of capillary-based separation techniques. Inaddition to capillary electrophoresis, the contiguous capillaries of thepresent invention may be used for capillary electrochomatographyseparations, as well as other microseparation techniques, examples ofwhich include capillary isoelectric focusing, capillaryisotachophoresis, and electrokinetic chromatography.

The electrospray sources of the present invention each include acontiguous capillary for separating and electrospraying a fluidcomprising analyte and electrolyte as described earlier. In onepreferred embodiment, the electrospray source 30 depicted in FIG. 2A andFIG. 2B has a support structure that has a substrate 32 having a channel34 in which the contiguous capillary 14 and conductive portion 16 isplaced. A reservoir 36 is affixed above the electrically conductiveportion 16 to prevent flexing of the capillary in the vicinity of theelectrically conductive portion, and to hold a buffer solutioncontaining electrolytes (not shown). An electrode 38 resides within thebuffer solution to provide an electrical contact with the electricallyconductive portion of the capillary. The electrically conductive portionis designed to provide a voltage along the capillary, to provide a sprayvoltage at the spray tip, or to provide both voltages.

In another embodiment, the electrospray source 40 depicted in FIG. 3Aand FIG. 3B has a support structure that comprises a reservoir 42 havingholes 44 through which the capillary passes and is affixed with asealing material, such as epoxy (not shown). The conductive portion 16resides within the reservoir 36 to prevent flexing of the capillary thatcould otherwise break the conductive portion, and to hold a buffersolution containing electrolytes (not shown). An electrode 38 resideswithin the buffer solution to provide an electrical contact with theelectrically conductive portion of the capillary. Accordingly, theelectrically conductive portion is designed to provide a voltage alongthe capillary, to provide a spray voltage at the spray tip, or toprovide both voltages.

The apparatuses provided by the present inventions are capable ofconveying analyte ions into an analytical instrument using thecontiguous capillaries and electrospray sources that are describedabove. In certain embodiments, each apparatus includes a contiguouscapillary having a electrically conductive portion near its spray tip,an electrode exterior to the electrically conductive portion, a spraycounter-electrode in proximity to the spray tip, and a power supplyconnected to the electrode and the spray counter-electrode to provide aspray voltage.

Also in each apparatus, the electrode exterior to the electricallyconductive portion is in electrically conductive contact with the fluidinterior to the electrically conductive portion of the capillary. Thespray counter-electrode being in proximity to the spray tip includes anopening in fluid communication with the analytical instrument. The powersupply, which is connected to the electrode and the spraycounter-electrode to provide a spray voltage for generating anelectrospray comprising analyte ions, conveys at least a portion of theanalyte ions through the opening and into the analytical instrument.

In one embodiment, each apparatus further includes a second electrodethat is in electrically conductive contact with the fluid upstream fromthe electrically conductive portion of the capillary. In thisembodiment, the apparatus further includes a second power supply toproduce an electrophoresis voltage between the electrode that iselectrically in contact with the conductive portion of the capillary andthe second electrode. In this embodiment, the second electrode is usedto effect electrophoresis separation of the fluid within the capillary.While any type of electrical contact may be provided between the secondelectrode and the fluid adjacent to the inlet end of the capillary,typically the type of contact is an electrolytically conductive contact,such as the placement of a suitable electrode directly in the fluid thatenters the inlet.

In another embodiment, the capillary further includes a secondelectrically conductive portion through which the second electrode is inelectrically conductive contact with the fluid. In this embodiment, thesecond electrically conductive portion may be provided as describedabove. Although the two electrically conductive portions may be similarin characteristics, they may also be different. The second electricallyconductive portion may be located upstream or downstream from the firstelectrically conductive portion. In certain embodiments, the secondelectrically conductive portion is typically in proximity to thecapillary inlet so that an electrophoresis voltage may be applied acrossthe conductive portions. In other embodiments, the second electricallyconductive portion resides between the spray tip and the firstelectrically conductive portion to provide a spray voltage.

The apparatuses of the present invention may be used to convey analyteions into any type of analytical instrument, typically the analyticalinstrument has an inlet for receiving fluid samples. In theseembodiments, the inlets for receiving fluid samples are typicallydesigned to place the sprayed analyte under vacuum. Analyticalinstruments that typically have an inlet for receiving samples includemass analyzers, such as mass spectrometers. In certain embodiments,analytical instruments may be coupled with the contiguous capillarieswithout placing the fluid samples under vacuum. In these embodiments,the spray tips may be placed in proximity to a nebulizing gas, such asin atomic absorption spectrophotometry.

The contiguous capillaries of the present invention may be made by themethods described herein. These methods include providing a capillaryand etching a portion of the capillary wall in proximity to the spraytip end. The spray tip may be formed during or after etching, but istypically formed before etching. The spray tip is typically formed byheating a section of the capillary at the spray tip end and drawing downthe heated capillary to form a narrowed opening. The etching provides anelectrically conductive portion of the capillary. In one embodiment ofthe present invention, the etching decreases (thins) the capillary wallthickness until the capillary wall at the etched area becomesconductive. In this embodiment, the etching does not penetrate thecapillary wall, thereby preventing the formation of pores that otherwisecould permit the passage of electrolyte molecules, analyte molecules, orboth.

In another embodiment, the etching penetrates a portion of the capillarywall to form pores. As described earlier in the description of theconductive portion of the capillary, and while not being bound to aparticular theory of operation of the conductive portions, it isbelieved that the pore sizes may be substantially smaller than at leasta portion of the fluid components, in which case at least a portion ofthe fluid components do not pass through the pores. In cases where atleast a portion of the fluid components are about the same size as thepores, then at least a portion of the fluid components may pass throughthe pores. In certain embodiments, the pores are of size to at leastpartially block passage of larger analyte molecules while permittingpassage of smaller electrolyte molecules.

In one embodiment of making the contiguous capillary tubes, the etchingprocess includes contacting the portion of the capillary with an etchingfluid that is capable of dissolving the capillary material. In thisembodiment, the etching fluid may be a solvent for the capillarymaterial. Suitable solvents for plastic capillaries is provided in anumber of plastic materials reference books, such as The PolymerHandbook, 3rd Ed., Brandrup and Immergut Eds., Wiley Interscience 1989.A typical etching fluid that is used with fused silica capillaries ishydrofluoric acid.

In one embodiment of the present invention, the etching position isselected by providing a capillary having a protective coating that iscapable of protecting the capillary from the etching fluid, and removinga portion of the protective coating to provide an exposed area that isetched when submersed in the etching fluid. In this embodiment, theprotective coating is suitably removed by chemical or mechanical means,such as by scraping away a portion of the protective coating.

In certain embodiments, the coating is removed completely around thecircumference of the capillary to provide a completely circum-etchedconductive portion. Typically, only a portion of the protective coatingis removed from around the circumference to provide a partiallycircum-etched conductive portion. In these cases, the protective coatingis typically removed from about 20 percent to about 50 percent aroundthe capillary. The resulting partially circum-etched conductive portionis typically less prone to breakage than a completely circum-etchedconductive portion.

In one embodiment of the present invention, the etching is terminated assoon as the portion of the capillary wall becomes electricallyconductive therethrough. Etching may be terminated by filling thecapillary with a suitable electrolyte, contacting on end of thecapillary with an electrode, placing a second electrode in the suitableetching solution with the capillary portion to be made electricallyconductive, applying a suitable voltage across the electrodes,monitoring the current, and stopping the etching after the currentincreases to a value greater than zero.

Any suitable electrolytic fluid may be used to fill the capillary inthis process, an example of which is formic acid. Platinum electrodesare suitable for submerging in hydrofluoric acid (HF) etching solution,which is also a good electrolyte in contact with the developingelectrically conductive portion of the capillary. A suitable voltage istypically about 5 kV, with the submerged electrode typically at ground;lower and higher voltages are possible, as well as positive and negativevoltages. The current through the capillary is typically 0.0 microampsuntil electrical conduction is achieved, at which point the currentquickly becomes greater than 0.0. Removing the etching solution andrinsing with a suitable liquid, such as water, typically stops theetching when the current is less than about 10 microamperes, andtypically less than about 7 microamperes. During the etching process,the thickness of the portion of the capillary wall typically decreases.Upon completion, the thickness of the portion of the capillary wall thatis exposed to the etching solution is typically less than about 50microns.

In one embodiment, stopping the etching as soon as the current becomesgreater than 0.0 and less than about 10 microamps is desirable forthinning the capillary wall sufficiently to provide electricalconduction and minimizing the formation of pores that penetratecompletely through the capillary wall. In other embodiments, while notbeing bound to a particular theory of operation, longer etching timesafter which the current has reached about 10 microamps or greater may beused for forming pores that are large enough for passing electrolyteions, yet small enough to block the passage of analytes. Although longeretching times may result in larger pores, excessive etching times mayeventually cause the formation of pores, capillary breakage, or both. Inanother embodiment, removing the etching solution and rinsing with asuitable liquid, such as water, may be used to stop the etching beforepore formation or breakage occurs.

In the method of making the contiguous capillary tubes of the presentinvention, one embodiment further includes protecting the capillary frombreakage. In this embodiment, the capillary portion is affixed within avessel, such as a reservoir, which contains the etching solution. Apreferred embodiment of protecting the capillary from breakage includesaffixing a capillary 14 to a substrate 32 and a reservoir 36 as depictedin FIG. 2A and FIG. 2B, which is further described in the Examplesbelow. Another embodiment of protecting the capillary from breakageincludes affixing a capillary 14 to a reservoir 42 as depicted in FIG.3A and FIG. 3B. In this embodiment, the capillary 14 is threaded throughopenings 44 in the reservoir 42, and the capillary is affixed to thereservoir using a suitable sealing material, e.g., epoxy (not shown).Suitable materials of construction for the substrate 32, and reservoirs36 and 42 are typically selected to withstand chemical attack from theetching solutions. For example, acrylic plastic materials are suitablefor use with HF etching solutions.

The present invention also provides methods of conveying a fluidcomprising analyte and electrolyte into an analytical instrument, suchas a mass analyzer or a mass spectrometer. These methods to conveyfluids include providing a contiguous capillary as described above,transporting the fluid through the contiguous capillary, providing anelectrode exterior to the electrically conductive portion of acontiguous capillary as described above, and providing a spraycounter-electrode in proximity to the spray tip. A spray voltage is thenapplied between the electrode and the spray counter-electrode to effectelectrospray ionization of the analyte exiting the spray tip, so that atleast a portion of the analyte enters the analytical instrument.Typically, the spray counterelectrode is grounded and a spray voltage ofat least about 1 kV is applied.

In these methods, the capillary includes an inlet end to supply a fluidcomprising analyte and electrolyte into the capillary, a spray tip tospray fluid out of the end of the capillary that is opposite the inletend, and an electrically conductive portion of the capillary inproximity to the spray tip. The electrically conductive portion providesa voltage along the capillary interior, at the spray tip, or both. Also,as the fluid is being transported through the contiguous capillary, theelectrically conductive portion is typically designed to minimizeanalyte loss through the wall while maintaining electrical conductivity.Optionally, as provided above, the conductive portion of the capillarymay contain pores of a size to block passage of analyte while permittingpassage of electrolyte therethrough.

Also in these methods, the electrode exterior to the electricallyconductive portion is in electrically conductive contact with the fluidinterior to the capillary. The spray counter-electrode being inproximity to the spray tip includes an opening in fluid communicationwith the analytical instrument. The power supply is connected to theelectrode and the spray counter-electrode to provide a spray voltage,which generates an electrospray comprising analyte ions. At least aportion of the analyte ions is conveyed through the opening in the spraycounter-electrode and into the analytical instrument. Suitable sprayvoltages are typically greater than about 500 kV, and are more typicallygreater than about 1 kV.

In one embodiment of the present invention, the method of conveying afluid comprising analyte and electrolyte into an analytical instrumentfurther includes providing a second electrode in electrically conductivecontact with fluid upstream from the electrically conductive portion. Inthis embodiment, a voltage is applied between the electrode and thesecond electrode to effect electrophoresis separation of the fluidwithin the capillary. Although the second electrode may be in electricalcontact with the fluid anywhere upstream from the first electricallyconductive portion, the second electrode is typically in a location thatis in contact with fluid that is proximate to the inlet end of thecapillary.

In certain embodiments for conveying a fluid comprising analyte andelectrolyte into an analytical instrument, the capillary may have one ormore additional electrically conductive portions through which thesecond electrode is in electrically conductive contact with the fluid.In this embodiment, the second electrically conductive portion can belocated upstream or downstream from the first electrically conductiveportion. When the second electrically conductive portion is upstream,electrodes in electrical contact with both conductive portions may beused to generate an electrophoresis voltage for effecting separation ofthe analytes along the length of the capillary. Likewise, when thesecond electrically conductive portion is downstream, electrodes inelectrical contact with both conductive portions may be used to generatea spray voltage for effecting electrospraying of the analytes along thelength of the capillary.

Suitable spray voltages for conveying the fluid into an analyticalinstrument are typically at least about 500 volts, although lowervoltages may be used less efficiently. Likewise, suitableelectrophoresis voltages for separating the analytes along the capillarytube are typically at least about 1000 volts, although lower voltagesmay be used less efficiently.

The present invention also provides methods of obtaining mass spectra ofanalytes. Referring to FIG. 4, in these methods, the mass spectra areobtained by conveying at least a portion of the analytes, as provided bythe above methods, into a mass spectrometer 60 through an opening in thecounterelectrode 62, and measuring m/z of the analyte ions within themass spectrometer 60 to provide the mass spectra. In this embodiment, anelectrospray source as depicted in FIG. 2A and FIG. 2B is mounted on aXYZ stage 50. Fluid comprising analyte and electrolyte are separated inthe contiguous capillary 10, which is shown attached to reservoir 36,which contains electrode 38 submersed in an electrolytic buffer 46. Theelectrode 38 is in electrolytically conductive contact with the fluidinterior to the electrically conductive portion of the capillary (notshown), through the buffer 46.

In another embodiment of the present invention, the method of obtainingmass spectra further includes providing a second electrode inelectrically conductive contact with fluid upstream from theelectrically conductive portion and applying a voltage between theelectrode and the second electrode to effect electrophoresis separationof the fluid within the capillary. In this embodiment, the mass spectraof individual analyte components are obtained, such as depicted in FIG.5. This embodiment of the present invention enables the high-resolutionidentification of analytes, such as depicted in FIG. 6, which isdescribed further in the Examples section.

Abbreviations and Terminology Used Herein

As used herein, the phrase “contiguous capillary” refers to a singlepiece of capillary tubing. CE, capillary electrophoresis; HPLC, highperformance liquid chromatography; FAB, fast atom bombardment; ESI,electrospray ionization; MS, mass spectrometry; i.d., inside diameter;o.d., outside diameter; “spray voltage” is synonymous with “spraypotential”; UV, ultraviolet; m/z mass-to-charge ratio; um, micron; μm,micron; mm, millimeter; cm, centimeter; kV, kilovolt; KV, kilovolt; V,volt; mA, milliamperes; %, percentage; M, molar; ° C., degrees celsius;mg, milligram; mL, milliliter; nL, nanoliter; s, seconds; psi, poundsper square inch; HF, hydrofluoric acid; mM, millimolar; S/N,signal-to-noise ratio; g/mol, grams per mole; mol. Wt., molecularweight; RNA, ribonucleic acid; DNA, deoxyribonucleic acid.

EXAMPLES

Contiguous capillaries. The following procedure describes thefabrication the contiguous capillary electrospray sources prepared fromcommercially available polyimide-coated fused silica capillaries(Polymicro Technologies, Phoenix, Ariz.). A spray tip was prepared byheating the capillary (75 cm long, 360 μm o.d., 50 μm i.d.) near thespray tip end with a microtorch and pulling it to draw down the insidediameter of the capillary to approximately 25 um. The polyimide coatingwas partially removed from a 3˜4 mm section of the capillary at adistance of 5 cm from the spray tip, to provide a partially (⅓)circum-etched capillary. The capillary was trimmed to a total length of60 cm, and was mounted on an electrospray assembly as shown in FIGS. 2Aand 2B. The assembly was constructed from a 4.5 cm×1.5 cm acrylicplastic substrate with a channel milled along its length having a depththat was slightly larger than the capillary diameter, and a 1.5 cm×1 cmi.d. acrylic plastic reservoir that was glued on top of the substrate.The fused silica capillary was threaded in the channel from one side andout the other side of the reservoir so as to position the exposedsection of the fused silica capillary inside the reservoir. Five minuteepoxy was applied to the outside of the reservoir around the capillaryto seal the two openings and to affix the capillary on the substrate.

HF etching of the exposed fused silica segment was conducted accordingto a procedure first reported by Hu et al. (15) and recently utilized byWei and Yeung (16), with modifications. The reservoir was filled with20% HF so as to cover the exposed fused silica section, and allowed toincubate in a ventilation hood at room temperature for five hours. Thecapillary was continuously monitored for conductivity (initially 0.0microamps) and the etching reaction was terminated as soon as electricalconductance was established through the capillary wall. Over the courseof the reaction (approximately 6 hours), the capillary wall thinned toabout 15-20 microns. This conductive portion created by HF etching wasstable and protected from breakage inside the reservoir, which alsoserved as the buffer reservoir for closing the CE circuit and providingthe spray voltage.

CE-MS. An ion trap mass spectrometer (LCQ-DECA XP, ThermoFinnigan, SanJose, Calif.) equipped with a nanoelectrospray ionization source wasused for all CE-MS experiments. The contiguous capillary assembly wasmounted on the nanoelectrospray source as shown in FIG. 2 and finepositioning of the spray tip was achieved by using the manufacturer'sXYZ stage attached to the nanoelectrospray assembly. The reservoir wasfilled with the same type of buffer used for the CE separation, and anelectrode that was immersed in the buffer surrounding the conductiveportion inside the reservoir supplied the spray voltage. For MS analysisthe spray voltage was adjusted between 2.4 and 2.9 kV for optimum spraystability, and the capillary temperature was 180° C. The instrument wasoperated in a data-dependent tandem MS mode in which each full-scan massspectrum was followed by a tandem MS scan of the most intense ionobserved in the previous scan. Normalized collision energy was set to38%. A P/ACE System MDQ CE instrument was used to conduct the CEseparations (Beclcman Coulter, Fullerton, Calif.). The MDQ instrumentwas configured to accept a windowless capillary cartridge, where theinlet side of the capillary was threaded into the detector end of thecartridge and the spray tip end extended to the outside of theinstrument. Both the MS and the CE instruments were controlled usingThermoFinnigan Xcalibur software (San Jose, Calif.).

Chemicals. Formic acid was obtained from Flulca Chemical Corp.(Milwaukee, Wis.), 48% HF was obtained from Aldrich (St. Louis, Mo.),acetonitrile (HPLC grade) was obtained from EM Science, Merck(Darmstadt, Germany), Five minute epoxy was obtained from Devcon(Riviera Beach, Calif.), and [Glu1]-fibrinopeptide B was purchased fromSigma (St. Louis, Mo.). Water for all uses was doubly distilled anddeionized using a NANOpure Diamond (TM) water system (BarnsteadInternational, Dubuque, Iowa).

Results and Discussion

One aspect of CE that makes it particularly suited for high-throughputproteomic investigations is the ability to conduct rapid separations ofcomplex mixtures. In addition, because the CE capillary is constructedusing contiguous fused silica, there is minimal opportunity forcontamination due to carry-over effects from separation to separation.Because there is no need to regenerate a stationary-phase as in HPLC,the time between concurrent CE experiments is much less than in the caseof HPLC-based separations.

The present inventions provide for coupling CE directly online with MSdetection, without the use of a sheath liquid, that is both reliable andrugged and provides for the highly sensitive analysis of a variety ofanalytes. CE is particularly well suited for the separation ofmacromolecules, such as peptides and proteins in acidic buffers, as CEcolumns are known to be stable over extended periods of time whenoperated under these conditions. FIG. 2 illustrates one contiguouscapillary design for which both CE separation and the electrospray maybe accomplished through the construction of a conductive portion byetching an exposed capillary segment with HF, which results in thethinning of the capillary wall, with minimal effects on the capillaryinterior. Several similar CE columns have been fabricated and have beenused, one continuously for more than two weeks (approximately 100 CE-MSruns) without any observable deterioration in either CE separation orelectrospray performance.

In the initial demonstrations of the present inventions, highlysensitive peptide separation and MS detection/identification wasaccomplished as demonstrated in FIGS. 5 and 6. Additional experimentalconditions used in obtaining the data shown in FIG. 5 were as follows:Column: contiguous bare fused silica capillary, 60 cm×360 mm o.d.×50 mmi.d., having a electrically conductive portion as described above;separation potential: 15 kV; observed CE current: 12 mA; temperature:22° C.; buffer: 20% acetonitrile in 500 mM formic acid, pH=2.2; sampleconcentration: 4 mM; injection time: 5 s at 1 psi (˜6.5 nL totalinjection volume). Other conditions were as in the experimental section.

Shown in FIG. 5A is the capillary electropherogram of 25 femtomoles of astandard peptide ([Glu1]-fibrinopeptide B) detected online by anion-trap mass spectrometer. Shown in FIG. 5B is the[M+2H]2+[Glu1]-fibrinopeptide B molecular ion (m/z 768.8) anddemonstrates the high signal to noise ratio (S/N greater than about 100)typically observed in these experiments conducted using the electrosprayionization sources of the present invention. The electrospray ionizationsource operates under nano-flow conditions, to provide higher S/N. Basedon the observed S/N and assuming a detection limit at a S/N of 5, thelower sensitivity limit is estimated to be approximately 900 attomolesoperating under current routine conditions. This representsapproximately a 100-fold improvement in the detection limit relative toanalysis of the same sample on an equivalent CE column employing asheath flow interface (results not shown). The viability of using CEcoupled online with tandem MS for the identification of peptides fromthe resulting fragment ion spectrum is illustrated by FIG. 5C. Spectralinformation from FIG. 5C was used to search a non-redundant proteindatabase (http://www.ncbi.nih.gov) using the program SEQUEST, whichresulted in the positive identification of [Glu1]-fibrinopeptide B witha Xcorr score of 4.8 (data not shown).

Shown in FIG. 6 is the separation of a mixture of nine bioactivepeptides (analytes) at a concentration of 1 mg/mL. The analytes were:1=bradykinin; 2=substance P; 3=bradykinin fragment 1-5;4=[arg]-vasopressin; 5=luteinizing hormone releasing hormone;6=bombesin; 7=leucine enkephalin; 8=methionine enkephalin; 9=oxytocin.Additional experimental details used to obtain these results were:Column: contiguous bare fused silica capillary, 60 cm×360 mm o.d.×75 mmi.d. having a electrically conductive portion as provided above;separation potential: 20 kV; observed CE current: 19 mA; buffer: 5%acetonitrile in 1 M acetic acid, pH=2.4; temperature: 22° C.; sampleconcentration: 1 mg/mL; injection time: 5 s at 1 psi (˜25 nL totalinjection volume). Other conditions as provided above.

The inset of FIG. 6 gives the corresponding separation of the samepeptide mixture at a concentration of 25 mg/mL, using similar CEconditions. The peptides in FIG. 6 were easily identified by their MSspectra. Studies are currently under investigation to further developthe routine use of CE-tandem MS for peptide/protein identification.

Example. A novel, rugged capillary electrophoresis-electrosprayionization (CE-ESI) interface, where the separation column and the spraytip are constructed from a single piece of a fused silica capillary isdescribed. ESI is accomplished by applying an electrical potentialthrough an easily prepared conductive portion across a 3˜4 mm length ofthe fused silica capillary. A stable electrospray is produced atnanoflow rates generated in the capillary by electrophoretic andelectroosmotic forces. The interface is particularly well suited for thedetection of sub-femtomole levels of proteins and peptides. Theruggedness of this interface was evident by the continuous operation ofthe same column for over a two-week period with no detectabledeterioration in separation or electrospray performance. Injection of 25femtomole of [Glu1]-fibrinopeptide B using the new device produced aCE-ESI-MS electropherogram with a signal to noise ratio of over 100. Amixture of nine bioactive peptides at a concentration of 1 mg/mL wassuccessfully separated and detected.

Interface Fabrication. Fused silica capillaries (Polymicro Technologies,Phoenix, Ariz.) were used to fabricate the sheathless interfacesaccording to the following procedure: Spray tips were made by applyingheat from a microtorch while pulling gently. The resulting long taperedtip is later trimmed to the desired tip inside diameter using a glasstube cutter. The polyimide coating was removed from a 3-4 mm section ofthe capillary at a distance of 5 cm from the spray tip end and thecapillary was trimmed to a total length of 60 cm. The capillary wasmounted on the porous junction assembly as shown in FIG. 7. The assemblywas constructed from a 4.5 cm×1.5 cm Plexiglas slide where a groove wasmilled along its length having a depth slightly larger than thecapillary's outside diameter, and a 1.5 cm×1 cm i.d. Plexiglas reservoirthat is glued on top of the slide. The fused silica capillary wasthreaded in the groove from one side and out the other side of thereservoir so as to position the exposed section of the fused silicacapillary inside the reservoir. 5 Minute Epoxy glue (Devcon, RivieraBeach, Calif.), was applied to the outside of the reservoir around thecapillary to seal the two holes and to pin the capillary on the slide.Hydrofluoric acid (HF) etching of the exposed fused silica segment wasconducted according to a procedure first reported by Hu et al. (Hu, S.;Wang, Z.-L.; Li, P.-B.; Cheng, J. K., Anal Chem., 1997, 69, 264) andrecently utilized by Wei and Yeung (Wei, W.; Yeung, E.S., Anal. Chem.,2002, 74, 3899), with modifications. The reservoir was filled with 20%HF so as to cover the exposed fused silica section and allowed toincubate in a hood for 5 hours at room temperature. The capillary wascontinuously monitored and the etching reaction was terminated as soonas electrical conductance was established through the already porouscapillary wall. Over the course of the reaction (approximately 6 hours),the capillary wall thins to about 15-20 μm. Although this porousjunction created by HF etching is fragile, it is durable since it isfirmly held inside the reservoir, which also serves as the bufferreservoir for closing the CE circuit and providing the spray voltage.

CE-MS. An ion trap mass spectrometer (LCQ-DECA XP, ThermoFinnigan, SanJose, Calif.) equipped with a nanoelectrospray ionization source wasused for all CE-MS experiments. The porous junction assembly was mountedon the nanoelectrospray source as shown in FIG. 8, and fine positioningof the spray tip was achieved by using the manufacturer's XYZ stage. Thereservoir was filled with the CE buffer and the spray voltage wassupplied by a platinum electrode immersed in the buffer surrounding theporous junction inside the reservoir. For MS analysis the spray voltagewas adjusted between 2.0 and 2.5 kV for optimum spray stability, and acapillary temperature of 180° C. was used for all experiments. Theinstrument was operated in a data-dependent tandem MS mode in which eachfull-scan mass spectrum was followed by a tandem MS scan of the mostintense ion observed in the previous scan. Normalized collision energywas set to 38%. A P/ACE System MDQ CE instrument was used to conduct theCE separations (Beckman Coulter, Fullerton, Calif.). The MDQ instrumentwas configured to accept a windowless capillary cartridge, where theinlet side of the capillary was threaded into the detector end of thecartridge and the spray tip end extended to the outside of theinstrument. Both the MS and the CE instruments were controlled usingThermoFinnigan Xcalibur software (San Jose, Calif.).

Chemicals. Formic acid and acetic acid were obtained from Fluka ChemicalCorp. (Milwaukee, Wis.), 48% HF was obtained from Aldrich (St. Louis,Mo.), acetonitrile (HPLC grade) was purchased from EM Science, Merck(Darmstadt, Germany), 5 Minute Epoxy was obtained from Devcon (RivieraBeach, Calif.), [Glu¹]-fibrinopeptide B, typsin, equine apomyoglobin,and the bioactive peptide mixture were purchased from Sigma (St. Louis,Mo.). Water for all uses was doubly distilled and deionized using aNANOpure diamond water system (Barnstead Internations, Dubuque, Iowa).

Protein Digestion. 60 mmoles of apomyoglobin were dissolved in 1 mL of100 mM NH₄HCO₃, pH 8.2, and digested with trypsin for 18 h at 37° C., ata protein-trypsin ratio of 50:1 (w/w). The digest was lyophilized todryness and resuspended in 11 mL of 10 mM HCl.

Results and Discussion

A device was developed for the direct online coupling of CE with MS thatprovides for the highly sensitive analysis of peptides and proteins. Thedevice offers advantages over existing CE-MS interfaces, including easeof fabrication, ruggedness, durability, and a true zero dead volumejunction between the separation column and the spray tip. One aspect ofCE that makes it particularly suited for high-throughput proteomicinvestigations is the ability to conduct fast and efficient separationsof peptides and protein digests, based upon differences in analytes'charge-to-size ratios. CE is well known for its highly selectiveseparations even though the peak capacity is less than that of LC. Inaddition, because the CE capillary is constructed using open tubularfused silica, there is minimal opportunity for contamination due tocarry-over effects from separation to separation. Since there is no needto reequilibrate the column as in LC, the time between consecutive CEexperiments is much less than in the case of LC-based separations withgradient elution.

One design of the sheathless CE-MS interface is illustrated in FIG. 7.Conducting both the CE separation and the ESI is the construction of aporous junction by etching an exposed capillary segment with HF,typically results in the thinning of the capillary wall enough togenerate electrical contact without affecting the capillary insidediameter of the capillary. The porous junction is often times fragile,but once it is made and protected inside the reservoir it proved to bedurable. Throughout our investigations we have fabricated several ofthese CE columns and each lasted for more than two weeks (eg.approximately 100 CE-MS runs) without any observable deterioration ineither CE separation or electrospray performance.

To demonstrate the sensitivity of this online sheathless CE design, weanalyzed a series of peptide mixtures. The electropherogram of 25femtomoles of a standard peptide ([Glu¹]-fibrinopeptide B) detectedonline by an ion-trap mass spectrometer is shown in FIG. 9A. The[M+2H]²⁺ [Glu¹]-fibrinopeptide B molecular ion (m/z 786.8) shown in FIG.9B demonstrates the high signal to noise ratio (S/N >100) typicallyobserved in these experiments conducted using the present sheathlessonline CE design. The interface operates under nano-flow conditions,that results in higher S/N. Based on the observed S/N, it is estimatedthat the lower sensitivity limit is approximately 900 attomoles(assuming a detection limit at a S/N=5). This represents approximately a100 fold improvement in detection limit relative to analysis of the samesample on an equivalent CE column employing a sheath flow interface(results not shown). The viability of using CE online with tandem MS forthe identification of peptides from the resulting fragment ion spectrumis illustrated by FIG. 9C. Information from this spectrum was used tosearch a non-redundant protein database (http://www.ncbi.nih.gov) usingthe program SEQUEST, which resulted in the positive identification of[Glu¹]-fibrinopeptide B with a Xcorr score of 4.8 (data not shown).

The separation of a mixture of 9 bioactive peptides at a concentrationof 3 μg/mL (injection =75 fmole) is shown in FIG. 10. The inset to thisfigure shows the corresponding separation of the same bioactive peptidemixture at a concentration of 25 μ/mL, using similar CE conditions withUV detection at 214 nm. Compared to UV detection, the relative peakheights vary considerably because of differences in the ionizationefficiency of different peptides. Also MS generated peaks are broader,which without being limited to a theory of operation, are due to lack oftemperature control in the CE-MS set-up and the slow rate of dataacquisition by the MS. The direct detection of MS, however, allowed allof the peptides within this mixture to be easily identified based ontheir MS spectra. The CE-MS analysis of a tryptic digest of apomyoglobinis shown in FIG. 11. In this analysis 100 fmoles of the digest wasinjected onto the CE column and the peptides were detected in adata-dependent MS/MS mode. The electropherogram for this tryptic digestis shown in FIG. 11A. The MS spectrum of the peptide highlighted with anarrow is shown in FIG. 11B. The singly and doubly charged species ofthis peptide were detected with very high S/N, particularly for thedoubly charged ion. The tandem MS spectrum of the doubly charged parention is shown in FIG. hiC. Analysis of this spectrum using SEQUESTidentified this peptide as HGTVVLTALGGILK [SEQ ID NO: 3]fromapomyoglobin with Xcorr score of 4.95. Indeed, an almost complete y ionseries was identifiable for this peptide from the resulting tandem MSspectrum. Table I lists peptides that were identified by tandem MS withhigh Xcorr. Table II gives a list of all tryptic peptides that wereidentified by matching the molar mass of ions from the MS spectra with alist of expected peptides generated from the putative protein sequence.

CE is particularly well suited for the separation of peptides andproteins in acidic buffers as CE columns are known to be stable overextended periods of time when operated under these conditions. Formicacid and acetic acid were tested as buffer constituents with and withoutthe addition of organic solvents, and the best performance in terms ofCE separations and MS sensitivity was obtained with 1 M acetic acid(pH=2.4) without any additives. This buffer appears to be the most idealfor this application because it produced steady electrospray and lowfemtomole sensitivity without the use of organic solvent additives.Formic acid buffers, on the other hand, resulted in higher current andless sensitive MS detection. At pH 2.4 all peptides are positivelycharged and move towards the cathode at the spray tip outlet. The flowrate through the column is generated by electroosmotic flow with someassistance from the electrospray process. Electroosmotic flow is verylow with acidic buffers and cannot be easily measured by CE-MS becauseneutral electroosmotic flow markers do not generate a signal, however,an estimate of the upper limit of the flow rate can be easily obtainedfrom FIG. 10. Oxytocin, the last eluting peptide in the figure, tookover 26 minutes to migrate a distance of 60 cm with a linear velocity of2.3 cm/min. This linear velocity translates to a volumetric flow rate ofabout 100 nL/min for a 75 μm i.d. column. This flow rate represents anupper limit because oxytocin migrates under electrophoretic as well aselectroosmotic forces, and electroosmotic flow may be much less thanthis upper limit.

The addition of organic solvents (acetonitrile or methanol), althoughbeneficial to the electrospray process, especially with wide spray tips(<30 μm), caused peak broadening and deterioration in the separation ofpeptides. Adjustment of buffer pH with ammonia or triethylamine causedthe formation of dominant peptide adducts. During this investigation weused 1M acetic acid and obtained stable electrospray with 75 μm and 50μm i.d. columns having spray tip diameters ranging from 8 to 50 μm.Small diameter tips (<10 μm) produced steady spray with low sprayvoltages (1-1.5 kV), but from an operational point of view, they areless desirable because they tend to cause excessive back pressure in theseparation column and clog easily. On the other hand, wide diameter tips(˜50 μm) are easier to maintain, yet, they are not less desirablebecause they produce inefficient ESI resulting in lower sensitivitymeasurements.

TABLE I Identified peptides from CE-MS/MS analysis of horse apomyoglobintryptic digest. Peptide Sequence Xcorr Fragment ions 1 HLKTEAEMK 2.9514/16 (SEQ ID NO: 1) 2 YLEFISDAIIHVLHSK 4.97 22/30 (SEQ ID NO: 2) 3HGTVVLTALGGILK 4.95 23/26 (SEQ ID NO: 3) 4 YKELGFQG 2.66 12/14 (SEQ IDNO: 4) 5 GLSDGEWQQVLNVWGK 4.63 21/30 (SEQ ID NO: 5)

TABLE II Identified peptides from CE-MS peptide mapping of horseapomyoglobin tryptic digest. Fragment Sequence Residues 1GLSDGEWQQVLNVWGK  1–16 (SEQ ID NO: 6) 2 VEADIAGHGQEVLIR 17–31 (SEQ IDNO: 7) 3 FDKFK 43–47 (SEQ ID NO: 8) 4 HLKTEAEMK 48–56 (SEQ ID NO: 9) 5HGTVVLTALGGILK 63–76 (SEQ ID NO: 10) 6 GHHEAELK 79–86 (SEQ ID NO: 11) 7PLAQSHATK 87–95 (SEQ ID NO: 12) 8 YLEFISDAIIHVLHSK 102–117 (SEQ ID NO:13) 9 HPGNFGADAQGAMTK 118–132 (SEQ ID NO: 14) 10 ALELFR 133–138 (SEQ IDNO: 15) 11 NDIAAK 139–144 (SEQ ID NO: 16) 12 YKELGFQG 145–153 (SEQ IDNO: 17)

1. An electrospray source, comprising: a contiguous capillary forseparating and electrospraying a fluid comprising analyte andelectrolyte, said contiguous capillary comprising: a spray tip at oneend of said capillary; and an electrically conductive portion of thecapillary in proximity to said spray tip, said electrically conductiveportion capable of blocking passage of analyte therethrough, wherein theelectrically cunductive portion comprises pores of a size that permitpassage of electrolyte therethrough.
 2. The electrospray source of claim1, wherein the electrically conductive portion is electrolyticallyconductive.
 3. The electrospray source of claim 1, wherein thecontiguous capillary comprises fused silica.
 4. The electrospray sourceof claim 1, wherein the spray tip has an opening smaller than about 50microns.
 5. The electrospray source of claim 1, wherein the pores permitpassage of electrolyte ions having a molecular mass of less than about300 g/mol.
 6. The electrospray source of claim 1, wherein the pores atleast partially block the passage of analyte.
 7. The electrospray sourceof claim 6, wherein the pores completely block the passage of analyteions having a molecular mass of greater than about 100 g/mol.
 8. Theelectrospray source of claim 1, wherein the electrically conductiveportion is affixed within a support structure, said support structurecapable of holding a buffer solution.
 9. The electrospray source ofclaim 1, wherein the electrically conductive portion of the capillarycomprises at least about 1 mm of the length of the capillary.
 10. Theelectrospray source of claim 1, wherein the electrically conductiveportion of the capillary has a wall thickness less than the wallthickness of the adjacent capillary portion.
 11. The electrospray sourceof claim 10, wherein the wall thickness of the electrically conductiveportion of the capillary is less than about 50 microns.
 12. Theelectrospray source of claim 1, wherein the diameter of the spray tipopening is smaller than inside diameter of the capillary.
 13. Acontiguous capillary for electrospraying a fluid comprising analyte andelectrolyte, the capillary comprising: an inlet end to supply fluid intothe capillary; a spray tip for spraying fluid out of the capillary; andan electrically conductive portion of the capillary in proximity to saidspray tip, said electrically conductive portion capable of blockingpassage of analyte therethrough, wherein the electrically conductiveportion comprises pores of a size that permit passage of electrolytetherethrough.
 14. The contiguous capillary of claim 13, wherein theelectrically conductive portion is electrolytically conductive.
 15. Thecontiguous capillary of claim 13, wherein the contiguous capillarycomprises fused silica.
 16. The contiguous capillary of claim 13,wherein the spray tip has a diameter opening of less than about 50microns.
 17. The contiguous capillary of claim 13, wherein the porespermit passage of electrolyte ions having a molecular mass of less thanabout 300 g/mol.
 18. The contiguous capillary of claim 13, wherein thepores at least partially block the passage of analyte.
 19. Thecontiguous capillary of claim 18, wherein the pores completely block thepassage of analyte ions having a molecular mass of greater than about300 g/mol.
 20. The contiguous capillary of claim 13, wherein theelectrically conductive portion is affixed within a support structure,said support structure capable of holding a buffer solution.
 21. Thecontiguous capillary of claim 13, wherein the electrically conductiveportion of the capillary comprises at least about 1 mm of the length ofthe capillary.
 22. The contiguous capillary of claim 13, wherein theelectrically conductive portion of the capillary has a wall thicknessless than the wall thickness of the adjacent capillary portion.
 23. Thecontiguous capillary of claim 13, wherein the wall thickness of theelectrically conductive portion of the capillary is less than about 50microns.
 24. The contiguous capillary of claim 13, wherein the diameterof the spray tip opening is smaller than the inside diameter of thecapillary.
 25. An apparatus for conveying analyte ions into ananalytical instrument, the apparatus comprising: a contiguous capillary,comprising: an inlet end to supply a fluid into the capillary, saidfluid comprising analyte and electrolyte; a spray tip to spray fluid outof the end of the capillary that is opposite to the inlet end; and anelectrically conductive portion of the capillary in proximity to saidspray tip, said electrically conductive portion capable of blockingpassage of analyte therethrough, wherein the electrically conductiveportion of the capillary comprises pores of a size that permit passageof electrolyte therethrough; an electrode exterior to said electricallyconductive portion, said electrode being in electrically conductivecontact with the fluid interior to said electrically conductive portion;a spray counter-electrode in proximity to said spray tip, said spraycounter-electrode comprising an opening in fluid communication with theanalytical instrument; and a power supply connected to the electrode andthe spray counter-electrode, said power supply providing a spray voltagefor generating an electrospray comprising analyte ions, whereby at leasta portion of the analyte ions are conveyed through said opening and intothe analytical instrument.
 26. The apparatus of claim 25, wherein theelectrically conductive portion of the capillary is electrolyticallyconductive.
 27. The apparatus of claim 25, further comprising: a secondelectrode in electrically conductive contact with fluid upstream fromthe electrically conductive portion of the capillary; and a second powersupply to produce an electrophoresis voltage between the electrode andsaid second electrode to effect electrophoresis separation of theanalytes within the capillary.
 28. The apparatus of claim 25, whereinthe second electrode is in electrolytically-conductive contact with thefluid adjacent to the inlet end of the capillary.
 29. The apparatus ofclaim 25, wherein the capillary further comprises a second electricallyconductive portion through which the second electrode is in electricallyconductive contact with the fluid, said second electrically conductiveportion being located upstream or downstream from the first electricallyconductive portion.
 30. The apparatus according to claim 25, wherein theanalytical instrument is a mass spectrometer or a mass analyzer.
 31. Acontiguous capillary, comprising: an inlet end to supply a fluid intothe capillary, said fluid comprising analyte; a spray tip for sprayingfluid out of the capillary; and an electrically conductive portion ofthe capillary in proximity to said spray tip, said electricallyconductive portion capable of blocking passage of analyte therethrough,wherein the electrically conductive portion comprises pores of a sizethat permit passage of electrolyte therethrough.
 32. The contiguouscapillary of claim 31, wherein the electrically conductive portion iselectrolytically conductive.
 33. The contiguous capillary of claim 31,wherein the contiguous capillary comprises fused silica.
 34. Thecontiguous capillary of claim 31, wherein the spray tip has a diameteropening of less than about 50 microns.
 35. The contiguous capillary ofclaim 31, wherein the pores permit passage of electrolyte ions having amolecular mass of less than about 300 g/mol.
 36. The contiguouscapillary of claim 31, wherein the pores at least partially block thepassage of analyte.
 37. The contiguous capillary of claim 36, whereinthe pores completely block the passage of analyte ions having amolecular mass of greater than about 300 g/mol.
 38. The contiguouscapillary of claim 31, wherein the electrically conductive portion isaffixed within a support structure, said support structure capable ofholding a buffer solution.
 39. The contiguous capillary of claim 31,wherein the electrically conductive portion of the capillary comprisesat least about 1 mm of the length of the capillary.
 40. The contiguouscapillary of claim 31, wherein the electrically conductive portion ofthe capillary has a wall thickness less than the wall thickness of theadjacent capillary portion.
 41. The contiguous capillary of claim 31,wherein the wall thickness of the electrically conductive portion of thecapillary is less than about 50 microns.
 42. The contiguous capillary ofclaim 31, wherein the diameter of the spray tip opening is smaller thanthe inside diameter of the capillary.
 43. The contiguous capillary ofclaim 31, wherein the electrically conductive portion extends about 20percent to about 50 percent around the circumference of the capillary.44. The contiguous capillary of claim 31, wherein the electricallyconductive portion extends completely around the circumference of thecapillary.